Purification of taxanes and taxane mixtures using polyethyleneimine-bonded resins

ABSTRACT

This invention is directed to a method for isolating and/or purifying one or more taxanes from materials comprising taxanes. In one embodiment, the method comprises the steps of: (a) treating a taxane containing material with an amino containing material attached to a solid matrix, such as a polyethyleneimine-bonded silica chromatographic resin; (b) eluting the desired taxane compound from the resin; and (c) recovering the eluted taxane compound.

I. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit under 35 U.S.C. §119(e) U.S. Provisional Application Serial Nos. 60/455,377 and60/456,478, filed Mar. 17, 2003, and Mar. 21, 2003, respectively. Eachof these applications is incorporated herein in its entirety.

II. FIELD OF INVENTION

[0002] The present invention relates to a process for separating orisolating one or more taxane compounds from materials containingtaxanes, and to the compositions or compounds resulting therefrom. Thepresent invention also relates to a process for purifying a biomassextract comprising one or more taxanes. In one embodiment, the processinvolves using one or more amino containing material attached to amatrix (e.g., solid matrix), including but not limited to,polyethyleneimine-bonded silica chromatographic resins (PBS resins).These resins facilitate the purification of taxanes in high yield andpurity. In one embodiment, the chromatographic resin comprises anon-functionalized and/or non-derivatized polyethyleneimine (“PEI”)polymer bonded to silica of various particle sizes or pore sizes. Inanother embodiment, the polyethyleneimine is methylated to form adiethylaminomethyl-bonded silica (“DEAM”). In one alternative embodimentof the present invention, both DEAM and PEI may be used in normal-phasechromatography under acidic pH.

III. BACKGROUND OF THE INVENTION

[0003] The present invention involves isolating or separating paclitaxelor other taxanes from materials containing one or more taxanes using anamino containing material supported by a solid matrix such as PBSresins. The present invention also involves purifying a biomass extractcomprising taxanes.

[0004] It is well known that various taxanes, e.g., paclitaxel, can bepurified by using chromatographic techniques. Chromatographic media suchas silica, alumina, alkyl-bonded silica resins such as C18 and C8, andpolystyrene divinylbenzene resins have been reported in the literatureto be useful for this purpose. However, depending on the nature of thepurification process, all of these media have limitations.

[0005] It has been discovered that amino containing material attached toa matrix (e.g., PBS resins) may provide superior selectivity andresolution over other conventional chromatographic resins used toisolate and purify taxanes. Therefore, amino containing materialsattached to a matrix (e.g., PBS resins) can be utilized to performseparations more efficiently than other resins in the area of taxanepurification. For instance, in the literature, the separation ofpaclitaxel (taxol A) and cephalomannine (taxol B) has been described asdifficult. The separation of these compounds is greatly facilitatedusing one or more PBS resins, as described herein. Also, since PBSresins can be utilized using organic solvents, the resin may be moreeasily loaded and samples can be applied at higher loading thanreverse-phase resins such as C18, C8, and polystyrene divinylbenzene.

[0006] PBS resins are primarily used in ion-exchange chromatographyapplications. Very few taxanes contain an ionizable group such ascarboxyclic acid or amine groups and therefore the mode of interactionis not traditional ionic exchange. Unexpectedly, the use of PBS resinsprovides superior selectivity and resolution in isolating or purifyingtaxanes. In the present invention, the PBS resins work best when smallamounts of an acidic modifier or salt are used in the mobile phase.

[0007] In addition, we have discovered that the amino groups of the PEIpolymer can be functionalized by alkylation, arylation, or acylation. Wehave also discovered that PBS resins may be used to perform preparativeand analytical separations of taxane compounds. Preparative separationscan be carried out in a batch, semi-continuous, or continuous mode.Semi-continuous mode may be in the form of simulated moving bed (SMB)chromatography.

IV. SUMMARY OF THE INVENTION

[0008] The present invention involves isolating or separating paclitaxelor other taxanes from materials containing one or more taxanes using anamino containing material attached (e.g., bonded) to a solid matrix,including but not limited to, a polyethyleneimine matrix capable ofisolating or separating taxanes, such as polyethyleneimine bonded tosilica. Other suitable amino containing materials attached to a matrix(e.g., solid matrix) may include those materials disclosed in U.S. Pat.Nos. 5,085,779 and 5,092,992. Both of these patents are incorporatedherein by reference in their entirety.

[0009] In one alternative embodiment, the present invention is directedto the use of PBS resins to aid in the separation or purification ofvarious taxanes, including paclitaxel, from compositions containingtaxanes. It is believed that the use of these resins for this purpose isboth novel and superior over the prior art. The preferred resins includePEI and DEAM.

[0010] In one alternative embodiment, the present invention is directedto a method of isolating one or more taxanes, and analogues thereof,from a taxane mixture. The taxane mixture may include a biomass extractor it may be obtained from semi-synthetic or total synthetic processes.The method comprising the steps of:

[0011] (a) treating the taxane mixture with an amino containing materialattached to a matrix (e.g., PBS resin);

[0012] (b) eluting one or more taxanes and their analogues from thechromatographic resin; and

[0013] (c) recovering the one or more taxanes and their analogues in oneor more fractions of eluate.

[0014] In another alternative embodiment, the present invention isdirected to a method of purifying and/or increasing the concentration oftaxanes in a taxane containing material, such as a Taxus extract, ortaxane mixture obtained from semi-synthetic or total syntheticprocesses, derived form plant material selected from the group of plantsknown as Yew. The method comprising the steps of:

[0015] (a) treating the material comprising taxanes and their naturalanalogues with an amino containing material attached to a matrix (e.g.,PBS resin);

[0016] (b) eluting the taxanes and their analogues from thechromatographic resin; and

[0017] (c) recovering the taxane and their analogues in one or morefractions of eluate.

[0018] The present invention and its advantages will be furtherunderstood by reference to the following detailed description and theaccompanying drawings.

V. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 shows an exemplary chemical reaction scheme using achromatographic resin of the present invention.

[0020]FIG. 2 shows an exemplary chemical reaction scheme using achromatographic resin of the present invention.

[0021]FIG. 3 shows separation of taxol A from taxol, B, C and otherimpurities using a PEI resin of the present invention.

[0022]FIG. 4 shows separation of semi-synthetic taxol A from othertaxane by-products using a DEAM resin of the present invention.

[0023]FIG. 5 shows non-limiting taxane molecules.

[0024]FIG. 6 show non-limiting, exemplary compounds (“primary taxanes”).

[0025]FIGS. 7-9 show non-limiting, exemplary taxane molecules.

[0026]FIG. 10 shows comparison of retention times and taxane standardson various media.

VI. DETAILED DESCRIPTION OF THE INVENTION

[0027] As used herein, an “alkoxy group” means a linear, branched, orcyclic saturated hydrocarbon attached to an oxygen atom. Preferably, analkoxy group has between one and six carbon atoms. An alkoxy group alsorefers to substituted alkoxy groups, which may include substituents suchas alkanoyloxy groups, alkenyl groups, alkyl groups, alkylsilyl groups,alkylsulfonyl groups, alkylsulfoxy groups, alkylthio groups, alkynylgroups, amino groups such as mono- and di-alkylamino groups and mono-and di-arylamino groups, amide groups, aryl groups, arylalkyl groups,carboxy groups, carboxyalkoxy groups, carboxyamide groups, carboxylategroups, haloalkyl groups, halogens, hydroxyl groups, nitrile groups,nitro groups, phosphate groups, siloxy groups, sulfate groups,sulfonamide groups, sulfonyloxy groups, and combinations of these.Preferred examples of alkoxy groups include, among others, methoxy,ethoxy, propoxy, cyclopropoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, tert-butoxy, cyclobutoxy, pentoxy, isopentoxy, neo-pentoxy,cyclopentoxy, hexoxy, and cyclohexoxy.

[0028] As used herein, an “alkyl group” means a linear, branched, orcyclic saturated hydrocarbon. Preferably, an alkyl group has between oneand six carbon atoms. An alkyl group also refers to substituted alkylgroups, which may include substituents such as alkanoyloxy groups,alkenyl groups, alky groups, alkylsilyl groups, alkylsulfonyl groups,alkylsulfoxy groups, alkylthio groups, alkynyl groups, amino groups suchas mono- and di-alkylamino groups and mono- and di-arylamino groups,amide groups, aryl groups, arylalkyl groups, carboxy groups,carboxyalkoxy groups, carboxyamide groups, carboxylate groups, haloalkylgroups, halogens, hydroxyl groups, nitrile groups, nitro groups,phosphate groups, siloxy groups, sulfate groups, sulfonamide groups,sulfonyloxy groups, and combinations of these. Preferred substituentsare alkoxy groups, amino groups such as dialkylamino groups, diarylaminogroups, carboxylic acid-containing groups, haloalkyl groups, halogens,hydroxyl groups, nitrile groups, nitro groups, and sulfonic acid groups.Examples of preferred alkyl groups include, but are not limited to,methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl, neo-pentyl,1-ethylpropyl, cyclopentyl, hexyl, and cyclohexyl.

[0029] As used herein, an “aryl group” means a phenyl group or naphthylgroup, which is optionally substituted. Examples of substituents on arylgroups include, but are not limited to, alkanoyloxy groups, alkenylgroups, alkoxy groups, alkylsilyl groups, alkylsulfonyl groups,alkylsulfoxy groups, alkylthio groups, alkynyl groups, amino groups suchas mono- and di-alkylamino groups and mono- and di-arylamino groups,amide groups, aryl groups, arylalkyl groups, carboxy groups,carboxyalkoxy groups, carboxyamide groups, carboxylate groups, haloalkylgroups, halogens, hydroxyl groups, nitrile groups, nitro groups,phosphate groups, siloxy groups, sulfate groups, sulfonamide groups,sulfonyloxy groups, and combinations of these. Preferred substituentsare alkoxy groups, alkyl groups, amino groups such as dialkylaminogroups and diarylamino groups, carboxylic acid-containing groups,haloalkyl groups, halogens, hydroxyl groups, nitrile groups, nitrogroups, and sulfonic acid groups.

[0030] As used herein, an “arylalkyl group” means an aryl group attachedto an alkyl group. An example of an arylalkyl group is a benzyl group.

[0031] As used herein, a “basic baccatin III structure” means a compoundhaving the formula as shown in FIG. 6, where each of R₁, R₂, R₄, R₇, R₁₀and R₁₃ independently is hydrogen, an alkyl group, an acyl group, anaryl group, an arylalkyl group, a vinyl group, an ether group, an estergroup, a glycoside group, an oxo group, or a hydroxyl protecting group.Included within the definition of a basic baccatin III structure isbaccatin III, which has the formula as shown in FIG. 7, and10-deacetylbaccatin III, which has the formula as shown in FIG. 8, whereAc is an acetyl or acetate group (CH₃C(O)—), and Bz is a benzoyl group(PhC(O)— or C₆H₅C(O)—).

[0032] As used herein, an “ester group” means a linear, branched, orcyclic substituent having an ester functionality, i.e., —C(O)—OR.Examples of ester groups include acyl groups such as aetyl and benzoyl,which are bound to a hydroxyl group.

[0033] As used herein, an “ether group” means a linear, branched, orcyclic substituent having an ether functionality, i.e., —C—O—C—. Anexamples of an ether group includes, but is not limited to,HOCH₂CH₂OC(CH₂OH)H—.

[0034] As used herein, a “glycoside group” or a “glycosyl group” meansany of a number of sugar derivatives that contain a non-sugar groupbonded to an oxygen or nitrogen atom and that on hydrolysis yield asugar such as glucose. An example of a preferred gylcosyl group isxylosyl.

[0035] As used herein, a “halogen” means fluorine, chlorine, bromine,and/or iodine.

[0036] As used herein, a “heterocyclic group” is a saturated,unsaturated, or aromatic cyclic compound that contains at least one atomother than carbon, e.g., oxygen, nitrogen, or sulfur, in a ring.Examples of heterocyclic groups include furyls such as 2-furan,morpholino, piperadino, piperazino, N-methylpiperazino, pyrrollyl,pyridyl, and thiophene.

[0037] As used herein, an “oxo-group” means a substituent derived fromthe oxidation of a glycoside group such as a xyloside as described inU.S. Pat. No. 5,356,928.

[0038] As used herein, “taxane or taxane molecule” includes but is notlimited to a molecule that contains a basic baccatin III structure witha (2R,3S)-C₆H₅CH(Rx)CH(OH)C(O)— group forming an ester with the hydroxylgroup located at the C-13 position of the basic baccatin III structure.The group represented by Rx can be an amino group, a salt of an aminogroup (e.g., an ammonium salt), an amino group which is protected withan amino protecting group, or a substituent which may be converted intoan amino group. Various isomers, homologues, and analogues of the basicbaccatin III structure, and of the (2R,3S)—C₆H₅CH(Rx)CH(OH)C(O)— groupalso are included in the definition of a taxane molecule. Also, a10-deacetylbaccatin III structure is contemplated within the scope of ataxane molecule. Included within the definition of a taxane or taxanemolecule include, but are not limited to, primary taxanes, for exampletaxol A (paclitaxel), taxol B (cephalomanninc), taxol C, taxol D, taxolE, taxol F, and taxol G. Further, the definitoin of a taxane or taxanemolecule includes docetaxel (TAXOTERE®). (See, e.g., FIGS. 5-6).

[0039] As used herein, a “vinyl group” means a linear or branchedsubstituent having a carbon-carbon double bond. Examples of vinyl groupsinclude, but are not limited to, 1-methyl-1-propenyl(CH₃CH═C(CH₃)—), and2-methyl-1-propenyl((CH₃)₂C═CH—).

[0040] Yew is a name ascribed to a number of trees which are Taxusspecies; Taxus being the main genus in the family Taxaceae. Originallyisolated from the bark of the Pacific yew (Taxus brevifolia) collectedfrom Washington State, beginning in 1962, taxol was subsequentlyreported as occurring in two other Taxus species, including Taxusbaccata (European yew) and Taxus cuspidata (Japanese yew), in 1971.Following intensive investigations, taxol was further reported to occurin a number of other Taxus species and cultivars. These include, but arenot limited to: Taxus globosa (Mexican yew), Taxus floridana (Floridayew), Taxus canadensis (Canadian yew), Taxus wallichiana (Himalayanyew), Taxus yunnanensis, Taxus chinensis, and also a number ofornamental hybrids, such as Taxus media cultivars, e.g.: T. media‘Densiformis’, T. media ‘Hicksii’, T. media ‘Brownii’, T. media ‘DarkGreen Spreader’, T. media ‘Runyan’, T. media ‘Wardii’, T. media‘Tautonii’, T. cuspidata ‘Capitata’, etc. In the present invention, theTaxus extract or a semi-synthetic reaction mixture may be derived fromany Taxus species, including but not limited to the species andcultivars described above. Other Taxus species for use in the presentinvention are identified in: Chadwick, L. C. and Keen, R. A. May 1986,“A study of the Genus Taxus”, Res. Bull. 1086, Ohio AgriculturalResearch and Development Center; Appendino, G. 1995, “The Phytochemistryof the Yew Tree”: Phytochemistry, Natural Products Reports 12(4):349-360; Convention On International Trade in Endangered Species of WildFauna and Flora: Eleventh meeting of the Plants Committee, LangKawi(Malaysia), 3-7 September 2001, Document PC11 DOC. 22-p.1, United Statesof America; and Greer, C. E., Schutzki, R. E., Fernandez, A. andHancock, J. F. October/December 1993. “Electrophoretic Characterizationof Taxus Cultivars”: HortTechnology, 3(4): 430-433. Each of thesereferences are incorporated herein by reference in their entirety.

[0041] It has now surprisingly been found that taxane compounds,including paclitaxel and paclitaxol analogs and congeners thereof can beisolated and purified from Taxus species in high yields by a normalphase liquid chromatography column packed with a PBS resin. By thesubject method, a large number of analogues of paclitaxel can beisolated from natural biomass extracts, and materials obtained fromsemi-synthetic or total synthetic processes.

[0042] The starting material for this invention may be a plant materialselected from the group of plants commonly referred to as yew trees. Themost suitable plants of this group are species of Taxus. Among Taxusspecies, Taxus x media cultivars are particularly preferred. Forexample, preferred cultivars include, but are not limited to, T. media‘Hicksii’ or T. media ‘Dark Green Spreader’. While it is convenient touse certain parts of the yew tree in this invention, taxol and itsnatural analogues can be extracted from the whole plant or fromseparated parts such as wood, stems, roots, leaves (needles), seeds, orany combination thereof. The material to be extracted can be eitherfresh or dried. Preferably, the bark or the needles are used. Further,the method of this invention can be used to purify taxanes from grownplant cells, or culture supernatants obtained by using in vitro culturetechnology. Additionally, the method is applicable to the separation andpurification of taxanes from mixtures treated by chromatographictechniques, or mixtures that have not been treated by such techniques.The method can be further applied to the separation and purification oftaxanes obtained from semi-synthesis or total synthesis procedures.

[0043] In one aspect of this invention, an amino containing materialattached to a matrix (e.g., silica) is used to separate or isolate oneor more taxane compounds from mixtures containing taxanes. Suchsubstances to be separated or purified include, but are not limited to,taxol A, B, C, D, E, F, G, Docetaxel, Nonataxel. Examples of such aminocontaining materials attached to a matrix include, but are not limitedto, PBS resins. These resins are typically used in ion-exchangeapplications, and therefore would not be expected to be useful forseparating neutral molecules, such as taxanes which generally contain noionizable groups. The selectivity and resolution of the separation oftaxane mixtures is surprisingly enhanced by using, for example, PBSresins. The PBS resins, for example, are more easily loaded to higherlevels than, for example, reverse-phase resins such as C18, C8 andpolystyrene divinylbenzene. PBS resins, for example, are alsoadvantageously used with organic solvents.

[0044] PBS resins may be derived from polyethyleneimine polymer bound tosilica of various pore sizes and/or particle sizes. The silica having anaverage pore size ranging from 60-300 Angstrom units. Preferably, thesilica has an average pore size of about 100 to 200 Angstrom units, morepreferably about 120 angstrom units. Also, the silica may have anaverage particle size ranging from about 0.25 to about 500 microns.Preferably, the silica has an average particle size from about 10-120microns, more preferably about 20 to 60, most preferably about 40.

[0045] A suitable PBS resin of the present invention may include DEAM.This resin may be purchased from J.T. Baker, CAS Reg. No. Product Codes:7317, 7471, 7472, 7473, under the name “BAKERBOND® DEAM ChromatographyPacking.” Another suitable PBS resin may include PEI. This resin mayalso be purchased from J.T. Baker, CAS Reg. No.: 126850-07-5, ProductCodes: 7134, 7180, 7264, 7368, 7476 and 8179, under the name“Polyethyleneimine Bonded Silica Gel.”

[0046] In one alternative embodiment of the present invention, the aminocontaining material attached to a matrix (e.g., PBS resin) may benon-derivatized, such as, for example, PEI. The chromatographic resinsof the present invention may include but are not limited to derivatizedPBS resins in which the primary and/or secondary amino groups of thepolyethyleneimine moiety are reacted with a reactive moiety. The PBSresins of the present invention may be represented by the generalizedformula:

Generalized Structure of PBS Resins

[0047]

[0048] The above structure is for illustrative, non-limiting purposesonly. The structure of the PBS resin may take on many other branchingpatterns or comprise single or multiple sites of attachment to thesurface of the silica particles. Also, the R groups may include, but arenot limited to, an H group, methyl group, an acyl group, alkyl group,aryl group, arylalkyl group, sulfonyl group, or any combination thereof.

[0049] The amino groups of the PEI polymer may be functionalized byalkylation, arylation, or acylation, or other means. Functionalizing theamino group may lead to a resin with increased selectivity, resolution,or other desired properties. For example, DEAM is a specific example ofa functionalized PEI.

[0050] The resins of the present invention may be used to isolate orpurify taxanes from a taxane mixture, including but not limited to, abiomass extract, such as a Taxus extract. In one alternative embodiment,the present invention may be used to isolate or purify taxanes fromtaxane mixtures (e.g., biomass extracts) produced by solventpartitioning, centrifugation, filtration, precipitation, or anycombination thereof. The resins of the present invention may also beused to isolate and/or purify taxanes from a naturally derived taxanecontaining mixture, wherein the taxanes were not formed from a syntheticor semi-synthetic process. Further, the resins of the present inventionmay be used to isolate or purify taxanes from mixtures comprisingtaxanes obtained from semi-synthetic or total synthetic processes.

[0051] It is well known that various forms of chromatography such assilica, alumina, C8, C18, polystyrene divinylbenzene, and others areuseful in purifying taxol A and other taxanes from Taxus extracts.Chromatography may be supplemented by other methods known to thoseskilled in the art, such as solvent partitioning and crystallization.PBS resins may be used in conjunction with these other techniques tosimplify the purification of taxol A and other taxanes from Taxusextracts. For example, we have discovered that PBS resins may beparticularly effective in separating taxol A from taxol B, N-methyltaxanes, taxane cinnamates, and others. PBS resins may also be used toseparate a related group of taxanes, such as primary taxanes (see FIG.6) from other taxanes found in Taxus extracts. The PBS resins may beused to separate taxanes from undesirable elements, thereby increasingthe taxane concentration in a mixture.

[0052] In one alternative embodiment, the starting material may compriseplant material selected from the group of plants commonly referred to asYew. The most suitable plants of this group are species of Taxus.Starting material for use in the present invention may include, but arenot limited to: (1) any material comprising one or more taxanes preparedfrom procedures other than semi-synthesis or total synthesis procedures;(2) any material comprising one or more taxanes prepared fromchromatography; (3) any material comprising one or more taxanes notprepared from chromatography; (4) any material comprising one or moretaxanes prepared by solvent partitioning, centrifugation, filtration,precipitation, or any combination thereof; (5) any material comprisingone or more taxanes from Taxus plants; or (6) any material comprisingone or more taxanes from one or more Taxus plants wherein the taxanesare not derived solely from Taxus brevifolia. The starting material mayinclude any material comprising one or more taxanes prepared by anycombination of the parameters set forth above. Methods of preparingtaxane containing materials (e.g., biomass extracts or semi-synthetic ortotal synthetic reaction mixtures) are known in the art.

[0053] In another alternative embodiment, the present invention isdirected to a method of isolating one or more taxanes from a taxanecontaining mixture, the method comprising the steps of: (a) treating themixture with a PBS resin; wherein the one or more taxanes are derivedfrom one or more Taxus plants, wherein the one or more taxanes are notderived solely from Taxus brevifolia; (b) eluting the one or moretaxanes from the PBS resin with an eluant; and (c) recovering the elutedone or more taxanes.

[0054] In another embodiment, the present invention is directed to amethod of isolating one or more taxanes from a taxane containingmixture, the method comprising the steps of: (a) treating the mixturewith a PBS resin; wherein the mixture comprises less than 25% or greaterthan 40% by weight of primary taxanes; (b) eluting the one or moretaxanes from the PBS resin; and (c) recovering the eluted one or moretaxanes.

[0055] In another embodiment, the present invention is directed to amethod of isolating one or more taxanes, the method comprising the stepsof: (a) treating a taxane containing mixture with a PBS resin; whereinthe mixture comprises from about 25% to 40% by weight of primarytaxanes; wherein the one or more taxanes are not derived solely fromTaxus brevifolia; (b) eluting the one or more taxanes from the PBSresin; and (c) recovering the eluted one or more taxanes.

[0056] In one alternative embodiment of the present invention, thestarting material may comprise less than about 25% or greater than about40% by weight of primary taxanes, including but not limited to taxol A,B, C, D, E, F or G. The remaining materials in the extract may compriseother compounds, including but not limited to impurities. Other suitableamounts of primary taxanes in the starting material may include fromabout 0.5% to 1%; 1% to 10%; 10% to 15%; 15% to 20%; 20% to 25%; 25% to30% or 30% to 35%, or 35% to 40%. Other amounts may also include about40% to 50%; 50% to 60%; 60% to 70%; 70% to 80%; 80% to 90% or 90% to100%.

[0057] In another alternative embodiment, the biomass extract is derivedfrom a Taxus plant. In yet another alternative embodiment, the biomassextract is derived from any Taxus plant, excluding Taxus brevifolia. Inyet another alternative embodiment, the biomass extract is derived fromany Taxus plant, excluding Taxus brevifolia, and comprises from about25% to about 40% by weight of primary taxanes, including but not limitedto taxol A, B, C, D, E, F or G.

[0058] In another embodiment, the present invention is directed to amethod of purifying one or more taxanes compounds from a biomassextract, wherein the biomass extract is derived from one or more Taxusplants. In one embodiment, the biomass extract is from one or more Taxusplants, excluding Taxus brevifolia. In another embodiment, the biomassextract comprises less than 25% or greater than 40% by weight primarytaxanes. In yet another embodiment, the biomass extract comprises fromabout 25% to about 40% primary taxanes, wherein the biomass extract isderived from one or more Taxus plants, excluding Taxus brevifolia.

[0059] In addition, in one alternative embodiment, the biomass extractmay comprise isobutyl alcohol in an amount less than about 50%, 40%, 30%or 20%, preferably less that 10%, more preferably less that 5%, mostpreferably less than 3%, 2%, 1%, 0.5%, or 0.25%.

[0060] The process of the present invention may increase the purity ofthe biomass extract by about 10% to 20%; 20% to 30%; 30% to 40%; 40% to50%; 50% to 60%; 60% to 70%; 70% to 80%; 80% to 90%; 90% to 100%; 100%to 110%; 110% to 120%; 120% to 130%; 130% to 140%; 140% to 150%; 150% to200%; 200% to 250%; 250% to 300%; 300% to 350%; 350% to 400%; 400% to450%; 450% to 500%; 500% to 550%; 550% to 600% 600% to 650%; 650% to700%; 700% to 750%; 750% to 800%; 800% to 850%; 850% to 900%; 900% to950%; 950% to 1000%. As used herein, the term purity means the weightpercent of one or more taxane compounds present in a dried form of thematerial or biomass extract.

[0061] The resin of the present invention may be used to purify one ormore taxanes from a taxane mixture obtained, in whole or in part, from asemi-synthetic or total synthetic process. In one alternativeembodiment, the resin of the present invention may be used to purifysemi-synthetic taxol A or other semi-synthetic taxanes from a crudereaction mixture. Because most synthetic reactions generate by-productsor unreacted starting materials that in many cases are structurallyclosely related to the desired product, the process of purifying suchproducts is very important. For example, PBS resins have been shown toenhance and simplify the purification of semi-synthetic taxol A producedfrom processes described herein from many related impurities andstructurally related compounds as shown in FIG. 10, for example.

[0062] In one alternative embodiment, the present invention involvespurifying one or more taxane compounds from materials prepared from asemi-synthesis or total synthesis process. FIGS. 1 and 2 show anexemplary, non-limiting semi-synthesis processes utilizing the resins ofthe present invention. The processes are described in Serial Appl. No.PCT/US03/10556 entitled “Conversion of Taxane Molecules and Serial Appl.No. US ______” Methods and Compositions for Converting Taxane Amides toPaclitaxel or Other Taxanes” filed Aug. 4, 2003.

[0063] In one alternative embodiment, such materials comprising lessthan about 10% by weight of C-2′ benzoyl primary taxanes, preferablyless than about 5%, more preferably less than about 3%, most preferablyless than about 1%. In another alternative embodiment, the taxanecontaining materials comprising less than about 9%, 8%, 7%, 6%, 4%, 2%,1%, 0.5%, 0.1%, or 0.01%. The taxane impurities include, but are notlimited to, the C-2′ benzoates of taxol A, B, C, D, E, F, or G. Thetaxane impurities may also include less than about 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1% or 0.5% of the C-2′ benzoates of taxol B, C, D, E, F,or G. As used herein, the term weight percent means the percentage ofone or more compounds present in a dried solid form of such material.

[0064] In one alternate embodiment, the present invention is directed toa method of isolating one or more taxanes from material comprisingtaxane compounds obtained from a semi-synthesis or total synthesisprocess, the method comprising the steps of: (a) treating the materialwith a PBS resin; wherein molecules used as reactants in thesemi-synthetic or total synthetic process are not derived solely fromTaxus brevifolia; (b) eluting the one or more taxanes from the PBSresin; and (c) recovering the eluted one or more taxanes.

[0065] In another embodiment, the present invention is directed to amethod of isolating one or more taxanes from material comprising taxanecompounds obtained from a semi-synthesis or total synthesis process: (a)treating the material with a PBS resin; wherein the material comprisesless than from about 8% to 3% by weight of 2′ benzoates of taxol A, B,C, D, E, F or G, combined; (b) eluting the one or more taxanes; and (c)recovering the eluted one or more taxanes. In another embodiment, thepresent invention is directed to a method of isolating one or moretaxanes from material comprising taxane compounds obtained from aprocess that excludes the step of benzoylating the C-2′ hydroxyl groupof the taxane molecules.

[0066] In another embodiment, the present invention is directed to amethod of isolating one or more taxanes from material comprising taxanecompounds obtained from a semi-synthesis or total synthesis process, themethod comprising the steps of: (a) treating the material with a PBSresin; wherein the material comprises less than 0.5% by weight of 2′benzoates of taxol B, C, D, E, F or G, combined; (b) eluting the one ormore taxanes; and (c) recovering the eluted one or more taxanes.

[0067] In another alternative embodiment, the present invention isdirected to a method of preparing a taxane mixture. The methodcomprising the steps of treating a material comprising one or moretaxanes obtained by a semi-synthetic or total synthetic process with aPBS resin. The taxanes or other compounds used in the semi-syntheticprocess are derived from one or more Taxus plants, or one or more Taxusplants excluding Taxus brevifolia. In another embodiment the taxanecontaining material comprising less than 3% by weight of 2′ benzoates oftaxol A, B, C, D, E, F or G. In another embodiment, the taxanecontaining material comprising less than 0.5% of the C-2′ benzoates oftaxol A, B, C, D, E, F or G.

[0068] In another alternative embodiment, materials to be processed bythe present invention may comprise benzoic anhydride, benzoic acid andbenzoyl chloride, preferably in amounts less than 10%, preferably lessthan 5%, more preferably less than 1% and most preferably less than0.3%.

[0069] With respect to materials obtained from semi-synthesis or totalsynthesis procedures, when these materials are processed by the presentinvention, the desired product(s) resulting therefrom may have a purityof at least 70%, preferably at least about 80%, more preferably at leastabout 90%, most preferably at least about 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100%.

[0070] In another alternative embodiment, the material to be processedby the present invention may comprise taxane impurities having amolecular weight of approximately 1104 and/or an oxetane ring-openedtaxanes having a molecular weights of approximately 871, among others.

[0071] The process of the present invention may increase the purity of aparticular taxane in the material by about 10% to 20%; 20% to 30%; 30%to 40%; 40% to 50%; 50% to 60%; 60% to 70%; 70% to 80%; 80% to 90%; 90%to 100%; 100% to 110%; 110% to 120%; 120% to 130%; 130% to 140%; 140% to150%; 150% to 200%; 200% to 250%; 250% to 300%; 300% to 350%; 350% to400%; 400% to 450%; 450% to 500%; 500% to 550%; 550% to 600% 600% to650%; 650% to 700%; 700% to 750%; 750% to 800%; 800% to 850%; 850% to900%; 900% to 950%; 950% to 1000%. As used herein, the term purity meansthe weight percent of one or more taxane compounds present in a driedform of the material.

[0072] One embodiment of the present invention involvespreparative-scale separations of taxane compounds, including paclitaxel,using a PBS resin. A preparative separation may be carried out in batch,semi-continuous, or continuous mode. Semi-continuous and continuousmodes may be in the form of simulated moving bed (SMB) chromatography.

[0073] In another embodiment, the present invention includes the use ofPBS resins to perform a separation by liquid chromatography. In one suchembodiment, a small amount of an acid or salt modifier is used in themobile phase of the liquid chromatographic separation. The acid or saltmodifiers used in the mobile phase may include, but are not limited to,acetic acid, formic acid, ammonium acetate or ammonium formate.

[0074] PBS resins may run in a normal-phase mode and exhibit uniqueselectivity for taxanes. For standard types of chromatography, both onC-18 and on silica, other taxanes can elute before and after paclitaxel.In normal-phase chromatography on PBS resins, most other structurallysimilar taxanes elute before paclitaxel allowing simplicity inpreparative separations. Of the most common taxanes, 10-deacetyltaxolelutes after paclitaxel on PBS resins. Fortuitously, the greatlyextended retention time of 10-deacetyltaxol allows for easy separationon preparative systems. This is important because 10-deacetyltaxol canbe a significant by-product in semi-synthetic preparations and is alsocommonly found in natural extracts of Taxus spp.

[0075]FIG. 10 shows comparison of retention times and taxane standardson various media. In FIG. 10, Footnote 1 represents Phenomenex™ Synergi™Hydro-RP, 4 mm (250×4.6 mm) HPLC column: acetonitrile/water gradientelution (40% to 60% ACN over 45 minutes) at 1.5 ml/min. Footnote 2represents Amicon Si-100-10 sp (250×4.6 mm) HPLC column: Isocraticelution, 60% ethyl acetate/40% hexanes and 1.0 ml/min. Footnote 3represents J.T. Baker® Wide-Pore PEI, 5 microns (250×4.6 mm) HPCCcolumn: Isocratic elution, 80% ethyl acetae/20% hexanes (with 0.5%Acetic Acid) at 1.0 ml/min. Footnote 4 represents J.T. Baker® Wide PoreDEAM, 5 micro (250×4.6 mm) HPCC column: Isocratic elution, ethyl acetate(with 0.5% acetic acid) at 1.0 ml/min. Footnote 5 represents retentiontimes from a single injection of a mixture of taxane standards. Footnote6 represents retention times relative to paclitaxel. Footnote 7represents times on an average of three injections of a single taxanestandard.

[0076] PEI bonded resins exhibit a similar selectivity for taxanes, butsome taxanes still elute in close proximity to paclitaxel (e.g.,10-deacetyl-7-epi-taxol, see FIG. 10). Chemically modified forms of PEI,such as DEAM, retain the same pattern of selectivity and exhibit greaterseparation of paclitaxel from its closest eluting taxanes.

[0077] The PBS resins have a high affinity for taxanes. As seen in FIG.10, a mixture of about 60% ethyl acetate and about 40% hexanes issufficient to elute paclitaxel in a reasonable time from silica. In somesituations, PEI requires a stronger mix of solvents: e.g., about 79.75%ethyl acetate, 19.75% hexanes and 0.5% acetic acid. DEAM's affinity forpaclitaxel was stronger than PEI's, requiring a mixture of about 99.5%ethyl acetate and about 0.5% acetic acid as its mobile-phase. Thestronger solvent systems used with PEI and DEAM dissolve taxanes to agreater extent thus allowing for higher loading.

[0078] In some variations, polyethyleneimine-bonded silica resins areused to separate various taxanes, including paclitaxel, from mixtures oftaxanes, such as Taxus extracts, or semi-synthetic or total synthetictaxane reaction mixtures. Suitable methods and compositions forproducing semi-synthetic taxane mixtures comprising paclitaxel weredescribed in Application Serial Nos. 60/401,191 (the '191 application),filed Aug. 4, 2002; PCT/US03/10557, filed Apr. 5, 2003; ProvisionalApplication entitled “Method and Compositions for Preparing aPharmaceutical Compound (e.g., Paclitaxel or other Taxanes) Using aBenzoylating Agent Essentially Free of Ring Chlorination”, U.S. Ser.Appl. No. 60/444,847 filed Feb. 4, 2003. Each of these applications isincorporated herein by reference in its entirety.

[0079] In one alternative embodiment, the process of the presentinvention comprises one or more of the following steps: (i) packing acolumn with an appropriate amount of PBS resin; (ii) equilibrating thecolumn with an organic solvent, preferably acetone containing aceticacid; (iii) loading a Taxus extract mixture or semi-synthetic or totalsynthetic reaction mixture onto the column; (iv) eluting the mixturewith an organic solvent, preferably acetone/acetic acid; (v) collectingthe eluate into one or more fractions; (iv) confirming the presence ofthe desired taxane in the one or more fractions; and (iv) purifying thedesired taxane by crystallization.

[0080] In accordance with one alternative embodiment, the taxanecontaining material is subjected to normal phase liquid chromatography(“NPLC”) in order to purify the taxol and other taxanes contained in acrude or semi-purified extract. Several variables are usually examinedto achieve separation and purification by liquid chromatography,including column packing (e.g., stationary phase or absorbent),composition of an eluant (e.g., mobile phase), column dimension, andeluant flow rate. These variables are known to those skilled in the artor can be readily determined without undue experimentation.

[0081] The chromatographic column dimensions, as well as thetemperature, flow rates, and time of chromatographic separations are notcritical to the practice of this invention, and are based primarily uponthe requirements for efficient chromatography which are known to thoseof skill in the art or can be readily determined without undueexperimentation.

[0082] In one alternative embodiment, chromatographic purification ofsemi-synthetic paclitaxel may be effected on DEAM resin (20 μmspherical, 100 Å) using ethyl acetate with 0.5% acetic acid as themobile phase. The progress of the separation may be monitored by UVabsorbance at an appropriate absorbing wave length, preferably 254 nmand 280 nm. The paclitaxel peak is collected in fractions. The ascendingportion (from baseline to apex) of the peak can be collected in severalfractions, typically 2 or 3. The earliest of these fractions willcontain most of the 7-epi-taxol and 10-deacetyl-7-epi-taxol impurities.The remaining portion of the peak may be collected in one or morefractions. A step-gradient of a 50:50 mixture of methanol and ethylacetate with 0.5% acetic acid may be employed to elute 10-deacetyltaxolfrom the column.

[0083] In one alternative embodiment, step gradient elution of theremaining impurities is preferred over use of a linear gradient forbrevity and ease of operation. A more complex equipment set is requiredto make use of a linear gradient. Step gradient elution, using apre-mixed solvent system, may be accomplished by changing a single valvesupplying the mobile phase to the pump. In another alternativeembodiment, it is preferred to delay introduction of methanol into themobile phase system because small amounts of methanol speed the elutionof a late eluting impurity, such 2-debenzoyltaxol, for example.

[0084] In one alternative embodiment, the liquid chromatography systemsof the present invention are preferentially used in a preparative mode(greater than 100 mg quantities). Preparative columns are typically 7 mmto 300 mm in diameter and 10 cm to 100 cm in length. Those skilled inthe art of chromatography can easily select a column with bed dimensionsappropriate to the amounts of material being purified. Flow rates of themobile phase are adjusted according to various factors including columndimensions, particle size and pore size of the resin, and desired peakresolution. Typical flow rates for preparative columns may range from 10ml/minute to 4 l/minute.

[0085] The times required for chromatographic runs range from about 10minutes to about 30 hours. Temperatures for chromatographic separationare typically at ambient temperature, although slightly highertemperatures can be used.

[0086] When practicing the chromatographic separation according to thepresent invention, the column may be operated in low-pressure (LPLC) tomedium-pressure (MPLC) modes, typically 10 to 500 p.s.i.g. It may alsobe run in high-pressure (HPLC) mode, typically 500 to 2000 p.s.i.g.

[0087] In another embodiment, the Taxus extract mixture orsemi-synthetic or total synthetic reaction mixture may be dissolvedprior to loading onto the column. For example, the mixture may bedissolved using an organic solvent, or by other means known to thosehaving ordinary skills in the art. A preferred organic solvent mixturecomprises acetone/acetic acid. The dissolved mixture may then be loadedonto a column packed with an appropriate amount of PBS resin.

[0088] Solvents (eluants) useful in this invention may be selected byreference to the standard practices of chromatography. Typically, amoderately polar organic solvent such as acetone, ethyl acetate,tetrahydrofuran, or acetonitrile may be used as eluant. Other ketones,ethers, and esters containing 1-5 carbons may be used as well. Modifiersto the eluant may include more polar solvents such as lower alcohols,acetic acid, and water if the mixture contains more polar taxanes, aswell as less polar organic solvents such as alkanes and halogenatedhydrocarbons if the mixture contains less polar taxanes. The percentagesof the modifiers may be 0-100% depending on the nature of the mixture tobe purified. This percentage can be readily determined by those skilledin the art.

[0089] In another embodiment, the Taxus extract mixture or crudereaction mixture may be dissolved in an organic solvent comprising ethylacetate/THF to form a taxane solution. The taxane solution may bestirred with mild heating, and then vacuum filtered to remove impuritiesand debris therefrom.

[0090] The crude taxane solution may also be diluted in an organicsolvent, preferably a EtOAc/THF solution. The solution may be injectedand/or loaded onto a column containing an appropriate amount of PBSresin. Beforehand, the column may be equilibrated with an organicsolvent, preferably EtOAc/acetic acid. After the taxane solution isinjected/loaded onto the column, EtOAc may be injected into the column.The eluted material may be collected in multiple fractions. A stepgradient comprising a methanol/EtOAc mixer solvent may be employed. Thedesired taxane is eluted from the column, and then collected in one ormore fractions. A wash step may be employed.

[0091] In some cases it may be advantageous to employ a gradient solventsystem, either step gradient or continuous gradient. The concentrationlimits of the gradients are determined by: (1) the concentration oforganic solvent necessary to elute taxanes from the absorbent; and (2)the requirement that the organic solvent be completely miscible andexist in a single phase at the concentration required to elute thetaxanes. For instance, 100% ethyl acetate may be used initially, thenswitching to 1-10% methanol in a single step, multiple step, orcontinuous gradient fashion. This system may be necessary to separatevarious taxanes that differ substantially in polarity and is readilydetermined by those skilled in the art.

[0092] The presence of the desired taxane in the one or more fractionsmay be detected using analytical techniques known in the art such asthin layer chromatography (TLC), infrared (IR) spectroscopy, nuclearmagnetic resonance (NMR) spectroscopy, high performance liquidchromatography (HPLC), reversed phase HPLC, and mass spectrometry (MS).

[0093] After the desired taxane(s) is collected from the column, it maybe further purified using other chromatographic methods, orcrystallization and/or one or more recrystallizations, depending on thedesired purity of the end product taxane molecule or taxane mixture.Crystallization and recrystallization may be conducted using a binary orternary solvent system, i.e., at least one solubilizing solvent and atleast one anti-solvent. Examples of solubilizing solvents include, amongothers, acetone, methyl-tert-butyl ether, methylene chloride, THF,methanol, ethanol, isopropyl alcohol, and acetonitrile. Examples ofanti-solvents include hydrocarbon solvents such as hexane and heptane,as well as water. In most cases, the solubilizing solvent and theanti-solvent are miscible in the ratios used. Examples of solventsystems useful with taxane molecules include, among others,acetone/hexane and methanol/water.

[0094]FIGS. 1 and 2 show an exemplary chemical reaction scheme using aPBS resin (e.g., DEAM) of the present invention. Specifically, it showsa semi-synthetic process of converting a taxane amide to paclitaxel orother taxanes, and purifying the taxane from the reaction product usinga PBS resin. Methods of converting a taxane amide to paclitaxel or othertaxanes are shown in the patent applications previously stated herein.

[0095] Having described specific chromatographic techniques andconditions suitable for this invention, a preferred embodiment of theisolation, separation, and purification of the taxane derivatives inaccordance with this invention is described below.

VII. EXAMPLES

[0096] The following examples are included for illustrative purposesonly and are not intended to limit the scope of the invention to anyparticular step or ingredient, for example.

Example 1

[0097] This example shows the use of PEI Resin in the Purification oftaxol A (Paclitaxel) and taxol B (Cephalomannine) from semi-purifiedTaxus media Densiformis extract. Here, a 75 liter column was packed with24.5 Kg of J.T. Baker PEI resin (40 micron particle size, 275 angstrompore size, J.T. Baker Item # 7264). The resin was equilibrated with 2column volumes, 150 liters, of acetone containing 0.5% acetic acid at aflow rate of 3.2 liters/min. The semi-purified Taxus extract feed wasthen loaded onto the column at a concentration of about 250 mg/mL inacetone and at a rate of 0.5 liters/min. The concentration of taxol was2.5 weight % or 2.5 grams of feed solid for 100 grams of PEI resin. Theweight percent of taxol A and taxol B in the feed solid was 10.8 weight%. The feed was eluted with acetone containing 0.5% acetic acid at aflow rate of 3.2 liters/min (superficial velocity=4.4 cm/min.). A totalof 10 fractions were collected. Fraction 1 and fraction 10 were 75liters each (1 column volume) and fractions 2-9 were 19 liters each (¼column volume). Product fractions 2-5 contained 87.96% of the totaltaxol A & B in the feed and the weight percentage of the combinedproduct fractions was 58.59 weight %. This product was adequate forfurther purification via crystallization.

Example 2

[0098] This example shows the use of PEI Resin in the Purification oftaxol A (Paclitaxel), taxol B (Cephalomannine), and taxol C fromimpurities found in semi-purified Taxus brevifolia extract. Here, solidfeed of semi-purified Taxus brevifolia extract was dissolved in acetonecontaining 0.6% acetic acid. This solution was loaded onto J.T. BakerPEI resin (40 micron particle size, 275 angstrom pore size, J.T. BakerItem # 7264). The feed load was 3% or 3 grams of feed solid for 100grams of PEI resin. The elution solvent was acetone containing 0.6%acetic acid. As the feed components were eluted from the column, sampleswere collected approximately every {fraction (1/20)}^(th) of a columnvolume. These fractions were analyzed by HPLC and the data was plottedas shown in FIG. 3. This run shows the separation of the impurities fromtaxol A, B, and C.

Example 3

[0099] This example illustrates the use of DEAM resin in thepurification of crude semi-synthetic taxol A (Paclitaxel). Here, themolecules used as reactants in the semi-synthetic or total syntheticprocess were derived from a Taxus plant, excluding Taxus brevifolia. Inthis example, the instrument used was a NovaPrep 200 Preparative HighPerformance Liquid Chromatography System combined with a Hitachi L-7400UV detector set to a wavelength of 254 nm. Both of these units aredirectly connected to a PC interface which runs the LC ReSpondercontroller software—Version 2.11.V (R & S Technology, Inc.).

[0100] The column is a Load & Lock 2” Preparative LC column (R & STechnologies, Inc./Varian) which has the following dimensions: InnerDiameter=5 cm; Length=25 cm; Volume=490.87 cm³. The column is packedwith Diethylaminomethyl (DEAM) Bonded Silica Gel: Spherical, 20 micronparticle size, 120 angstrom average pore size. The column is packed with270 g of resin compressed to 800 psi.

[0101] A total of 7.1 grams of crude taxol A was prepared via theprimary amine conversion chemistry described in U.S. Serial Appl. No.PCT/US03/10557 entitled “Conversion of Taxane Molecules.” The crudetaxol was dissolved using about 35 mL of solvent comprising of 90%(vol.) ethyl acetate/10% (vol.) tetrahydrofuran. The solution wasstirred with mild heating (˜35-40° C.) for 30 minutes. The clearsolution was vacuum filtered to remove any small fibers or particulatesbefore injection. After filtration, the solution was transferred to agraduated cylinder and diluted to a final volume of 42.5 mL (6×dilution) using the 90:10 EtOAc/THF solution. It was then remixed toensure uniformity before sampling. The loading for this run was 2.625%or 2.625 grams of feed solids for 100 grams of DEAM resin. The flow ratewas set at 90 mL/minute. The column was equilibrated for a period of 20minutes with the standard mobile phase consisting of EtOAc+0.5% aceticacid (v/v). At the 20-minute mark, the entire volume of the previouslyprepared sample solution was injected on the column immediately followedby a 20 mL injection line flush with EtOAc. The fractions were collectedin the following manner (all times listed are from injection point—time0): TABLE 2 Mass of Pac Fraction Open Close Total Time Area % Pac(mg) 1.  6:15 15:00  8:45 10.73  12.2 2. 15:00 15:15  0:15 74.95  96.43. 15:15 16:30  1:15 95.43  995.0 4. 16:30 29:00 12:30 99.25 4284.6 5.29:00 35:15  6:15 100.00  555.3 TOTAL: 5943.4 mg

[0102] A 50% step gradient was started at the 29 minute mark (i.e. frominjection point—time 0) consisting of a 50:50 methanol/ethyl acetatewash solvent mobile phase. This was run for 20 minutes total at whichpoint the run was complete. This wash step allows for faster elution ofthe 10-Deacetyltaxol A compound that elutes after taxol A. The lastfraction collection valve (i.e. fraction 5) was closed as soon as the UVdetector registered a rapid increase in absorbance, which indicateselution of the 10-Deacetyltaxol A. All of the 10-Deacetyltaxol A flushesfrom the column under the above condition within 10 minutes. However, toensure the column has been purged of any residual material and as aregular practice, the wash step is always a full 20 minutes in length.Thus, the entire run from the point of injection through the completecolumn wash step was 49 minutes. A trace of the chromatogram showing thecollection points of fractions 1-5 is shown in FIG. 4.

[0103] Fractions 3, 4, and 5 were combined in the pass pool forsubsequent crystallization work. The fraction analysis (by HPLC) of thepassing fractions gives a total of 5.835 g of taxol A. Dividing by thetotal mass of taxol in all fractions, the recovery is calculated to be98.18%. The purity of the crude material going onto the column was 87.9wt. % taxol, and after column purification, the purity of the passingcombined fractions was 98.71% area (by HPLC) taxol A.

Example 4

[0104] A 44 liter column was packed with 10 kg of J.T. Baker PEI resin(40 micron particle size, 275 angstrom pore size). The resin wasequilibrated with 3 column volumes, 135 liters, of 50 v %/50 v % ethylacetate and heptane containing 0.5% acetic acid at a flow rate of 1.0liter/min. The flow rate for the remaining fractions was at 1.0liter/min. The semi-purified feed (derived from Taxus media ‘Runyan’)was loaded onto the column at a concentration of 250 mg/ml in ethylacetate. The primary taxanes purity of the feed is 57 wt %. First, thefirst fraction was 170 liters of 50 v %/50 v % ethyl acetate and heptanecontaining 0.5 v % acetic acid. Second, three 10-liter fractions of 50 v%/50 v % ethyl acetate and heptane containing 0.5% acetic acid wascollected. Third, the product fraction was 90 liters of 90 v %/10 v %ethyl acetate and methanol. Lastly, the column was wash with 45 litersof 90 v %/10 v % ethyl acetate/methanol. The product pool contains 93.5wt % primary taxanes. The percent recovery of taxol A and B was 95%.

[0105] Throughout the description, where the present invention isdescribed as having, including, or comprising specific components, orwhere processes are described as having, including, or comprisingspecific process steps, it is contemplated that the present inventionalso consists essentially of, or consists of, the recited components orprocessing steps. Further, it should be understood that the order ofsteps or order for performing certain actions are immaterial so long asthe invention remains operable. Moreover, two or more steps or actionsmay be conducted simultaneously so long as the invention remainsoperable. Also, one or more steps or elements may be omitted from theclaimed invention, or the invention described herein suitably may bepracticed in the absence of any component or step which is notspecifically disclosed herein, so long as the invention remainsoperable.

[0106] Further, the present invention may be embodied in other specificforms without departing from the spirit or essential characteristicsthereof. The foregoing embodiments are therefore to be consideredillustrative rather than limiting the invention described herein.

[0107] The content of each patent and non-patent document referred toherein is expressly incorporated herein by reference in its entirety.

We claim:
 1. A method of isolating one or more taxanes from a taxanecontaining mixture, the method comprising the steps of: (a) treating themixture with a PBS resin; wherein the one or more taxanes are derivedfrom one or more Taxus plants, wherein the one or more taxanes are notderived solely from Taxus brevifolia; (b) eluting the one or moretaxanes from the PBS resin with an eluant; and (c) recovering the elutedone or more taxanes.
 2. A method of isolating one or more taxanes from ataxane containing mixture, the method comprising the steps of: (a)treating the mixture with a PBS resin; wherein the mixture comprisesless than 25% or greater than 40% by weight of primary taxanes; (b)eluting the one or more taxanes from the PBS resin; and (c) recoveringthe eluted one or more taxanes.
 3. A method of isolating one or moretaxanes, the method comprising the steps of: (a) treating a taxanecontaining mixture with a PBS resin; wherein the mixture comprises fromabout 25% to 40% by weight of primary taxanes; wherein the one or moretaxanes are not derived solely from Taxus brevifolia; (b) eluting theone or more taxanes from the PBS resin; and (c) recovering the elutedone or more taxanes.
 4. A method of isolating one or more taxanes frommaterial comprising taxane compounds obtained from a semi-synthesis ortotal synthesis process, the method comprising the steps of: (a)treating the material with a PBS resin; wherein molecules used asreactants in the semi-synthetic or total synthetic process are notderived solely from Taxus brevifolia; (b) eluting the one or moretaxanes from the PBS resin; and (c) recovering the eluted one or moretaxanes.
 5. A method of isolating one or more taxanes from materialcomprising taxane compounds obtained from a semi-synthesis or totalsynthesis process: (a) treating the material with a PBS resin; whereinthe material comprises less than about 8% by weight of C-2′ benzoates oftaxol A, B, C, D, E, F or G, combined; (b) eluting the one or moretaxanes; and (c) recovering the eluted one or more taxanes.
 6. A methodof isolating one or more taxanes from material comprising taxanecompounds obtained from a semi-synthesis or total synthesis process: (a)treating the material with a PBS resin; wherein the material comprisesless than 1.0% by weight of C-2′ benzoates of taxol B, C, D, E, F, or G,combined; (b) eluting the one or more taxanes; and (c) recovering theeluted one or more taxanes.
 7. A method of isolating taxol A from anaturally derived taxane containing mixture, said method comprising thesteps of: (a) treating the taxane mixture with a PBS resin; (b) elutingthe taxol A from the PBS resin; and (c) recovering the eluted taxol A.8. A method of purifying one or more taxanes from a biomass extract,said method comprising the step of: (a) preparing the biomass extract bymeans other than chromatography; (b) treating the biomass extract with aPBS resin; (c) eluting the one or more taxanes from the PBS resin; and(d) recovering the eluted one or more taxanes.
 9. The method of claims1-3, wherein the taxane containing material comprises a biomass extractderived from Taxus media cultivars,
 10. The method of claims 1-3,wherein the taxane containing material comprises a biomass extractderived from Taxus media ‘Hicksii’.
 11. The method of claims 1-3,wherein the taxane containing material comprises a biomass extractderived from Taxus media ‘Dark Green Spreader’.
 12. The method of claims1-3, wherein the taxane containing material comprises a biomass extractderived from Taxus baccata.
 13. The method of claims 1-3, wherein thetaxane containing material comprises a biomass extract derived fromTaxus cuspidata.
 14. The method of claims 1-3, wherein the taxanecontaining material comprises a biomass extract derived from Taxusfloridana.
 15. The method of claims 1-3, wherein the taxane containingmaterial comprises a biomass extract derived from Taxus canadensis. 16.The method of claims 1-3, wherein the taxane containing materialcomprises a biomass extract derived from Taxus wallichiana.
 17. Themethod of claims 1-3, wherein the taxane containing material comprises abiomass extract derived from Taxus yunnanensis.
 18. The method of claims1-3, wherein the taxane containing material comprises a biomass extractderived from Taxus chinensis.
 19. The method of claims 1-3, wherein thetaxane containing material comprises a biomass extract derived fromTaxus media ‘Densiformis’.
 20. The method of claims 1-3, wherein thetaxane containing material comprises a biomass extract derived fromTaxus media ‘Brownii’.
 21. The method of claims 1-3, wherein the taxanecontaining material comprises a biomass extract derived from Taxus media‘Hicksii’.
 22. The method of claims 1-3, wherein the taxane containingmaterial comprises a biomass extract derived from Taxus media ‘Runyan’.23. The method of claims 1-3, wherein the taxane containing materialcomprises a biomass extract derived from Taxus media ‘Wardii’.
 24. Themethod of claims 1-3, wherein the taxane containing material comprises abiomass extract derived from Taxus media ‘Tautonii’.
 25. The method ofclaims 4-6, wherein the taxane to be isolated is taxol A.
 26. The methodof claims 4-6, wherein the taxane to be isolated is taxol B.
 27. Themethod of claims 4-6, wherein the taxane to be isolated is taxol C. 28.The method of claims 4-6, wherein the taxane to be isolated is taxol D.29. The method of claims 4-6, wherein the taxane to be isolated is taxolE.
 30. The method of claims 4-6, wherein the taxane to be isolated istaxol F.
 31. The method of claims 4-6, wherein the taxane to be isolatedis taxol G.
 32. The method of claims 4-6, wherein the taxane to beisolated is docetaxel.
 33. The method of claims 1-8, wherein the PBSresin has an average pore size ranging from about 60 to about 300Angstrom Units.
 34. The method of claims 1-8, wherein the PBS has anaverage pore size ranging from about 100 to about 200 Angstrom Units.35. The method of claims 1-8, wherein the PBS has an average pore sizefrom about 120 Angstrom Units.
 36. The method of claims 1-8, wherein thePBS has an average particle size ranging from about 0.25 to about 500microns.
 37. The method of claims 1-8, wherein the PBS resin has anaverage particle size ranging from 1 to 100 microns.
 38. The method ofclaims 1-8, wherein the PBS has an average particle size ranging fromabout 10 to about 120 microns.
 39. The method of claims 1-8, wherein thePBS has an average particle size of about 20 to about 60 microns. 40.The method of claims 1-8, wherein the PBS has an average particle sizeof about 40 microns.
 41. The method of claims 1-8, wherein the PBS resinis DEAM.
 42. The method of claims 1-8, wherein the PBS resin is PEI. 43.The method of claims 1-8, wherein the PBS resin has an average pore sizeranging from about 60 to about 800 Angstrom Units.
 44. The method ofclaims 1-8, wherein the PBS resin has a primary or secondary amino groupon the polyethyleneimine moiety.
 45. The method of claims 1-8, whereinthe amino groups of the PEI polymer are functionalized.
 46. The methodof claims 4-6, wherein the eluted one or more taxanes have a purity ofat least about 70%.
 47. The method of claims 4-6, wherein the eluted oneor more taxanes have a purity of at least about 80%.
 48. The method ofclaims 4-6, wherein the eluted one or more taxanes have a purity of atleast about 90%.